Mast cells stimulated with antigen undergo extensive changes in their cytoskeleton. In the present study, we assess the impact of actin-modifying drugs and report that, in the presence of cytochalasin D, mast cells stop membrane ruffling, but instead bleb. Bleb formation is reversible following washout of cytochalasin D and occurs in an actin-polymerization-dependent manner. Bleb formation is inhibited by expression of constitutively active ezrin-T567D. Blebbing is also inhibited by blebbistatin, a myosin II inhibitor, implying myosin II activation in the process. We used a selection of inhibitors and observed that myosin II activation is dependent mainly on Ca2+–calmodulin, with only a small contribution from Rho kinase. The signalling pathways stimulated by antigen include PLC (phospholipase C) and PLD (phospholipase D). Bleb formation was dependent on activation of PLC, but not PLD. Primary alcohols, used previously as a means to reduce PLD-derived phosphatidic acid, were potent inhibitors of membrane blebbing, but a more selective inhibitor of PLD, FIPI (5-fluoro-2-indolyl des-chlorohalopemide), was without effect. FIPI also did not inhibit membrane ruffling or degranulation of mast cells, indicating that inhibition by primary alcohols works through an unidentified mechanism rather than via diversion of PLD activity as assumed. We also examined the requirement for ARF6 (ADP-ribosylation factor 6) and observed that its expression led to an increase in bleb size and a further increase was observed with the dominant-active mutant, ARF6-Q67L. Since ARF6-T27N had no effect on bleb size, we conclude that ARF6 needs to be active to regulate the size of the blebs.
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Research Article|
December 14 2009
Reversible bleb formation in mast cells stimulated with antigen is Ca2+/calmodulin-dependent and bleb size is regulated by ARF6
Yuhki Yanase;
Yuhki Yanase
1
*Lipid Signalling Group, Department of Neuroscience, Physiology and Pharmacology, University College London, University Street, London WC1E 6JJ, U.K.
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Nicolas Carvou;
Nicolas Carvou
1
*Lipid Signalling Group, Department of Neuroscience, Physiology and Pharmacology, University College London, University Street, London WC1E 6JJ, U.K.
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Michael A. Frohman;
Michael A. Frohman
†Department of Pharmacology and Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794, U.S.A.
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Shamshad Cockcroft
Shamshad Cockcroft
2
*Lipid Signalling Group, Department of Neuroscience, Physiology and Pharmacology, University College London, University Street, London WC1E 6JJ, U.K.
2To whom correspondence should be addressed (email s.cockcroft@ucl.ac.uk).
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Publisher: Portland Press Ltd
Received:
July 23 2009
Revision Received:
September 21 2009
Accepted:
October 21 2009
Accepted Manuscript online:
October 21 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 425 (1): 179–193.
Article history
Received:
July 23 2009
Revision Received:
September 21 2009
Accepted:
October 21 2009
Accepted Manuscript online:
October 21 2009
Citation
Yuhki Yanase, Nicolas Carvou, Michael A. Frohman, Shamshad Cockcroft; Reversible bleb formation in mast cells stimulated with antigen is Ca2+/calmodulin-dependent and bleb size is regulated by ARF6. Biochem J 1 January 2010; 425 (1): 179–193. doi: https://doi.org/10.1042/BJ20091122
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